Mosquito-borne diseases, including malaria, are the number-one killers of humans worldwide. A major obstacle in controlling these diseases is that mosquitoes have developed resistance to insecticides. The long-term goals of the proposed research are to determine molecular mechanisms that control the mosquito's response to insecticides and to use this information to develop novel, more effective therapies to prevent resistance development, to control resistant mosquitoes and, ultimately, to reduce mosquito-borne diseases and their impact. A large number of studies showed that point mutations of sodium channels reduce sodium channel sensitivity to pyrethroid insecticides and that, in turn, such mutations confer pyrethroid resistance. Despite such findings, there is still no comprehensive, global picture of the mutations involved in an entire sodium channel gene that contributes to insecticide resistance in any insect, including the mosquito. This proposal is seeking an answer to the question: How many mutations, both synonymous and nonsynonymous, in an entire sodium channel are involved in insecticide resistance in the mosquito? Preliminary research has isolated cDNAs of the full-length sodium channels from Culex quinquefasciatus S- Lab and the HAmCqG0, HAmCqG8, MoAmCqG0, and MoAmCqG8 mosquitoes, and 25 mutations (7 nonsynonymous and 18 synonymous) have been identified. The objective of the proposed research is to characterize the 25 sodium channel mutations in 7 mosquito strains, in 2 mosquito lines, and in 16 groups of mosquitoes that will be treated with different concentrations of permethrin. This characterization will determine which mutations or mutation combinations are involved in pyrethroid insecticide. Our hypotheses are that: (a) mutations involved in pyrethroid insecticide resistance ("key mutations") are prevalent in mosquito populations following insecticide selection and (b) the "key" mutation prevalence is related to the level of insecticide resistance. We predict that the introduction of the mutations or mutation combinations into the wild (susceptible)-type mosquito sodium channel will decrease the sensitivity of the sodium channel, in response to pyrethroid treatment. Our specific aims are to: 1) Define the relationship between the level of insecticide resistance, and the prevalence of sodium channel mutations and mutation combinations, 2) Determine the effects of key mutations on sodium channel sensitivity to pyrethroids, and 3) Determine the relationship between the allelic expression of key mutations in the mosquito sodium channel at the DNA level and at the RNA level. The proposed research uniquely focuses on all of the mutations that naturally occur in an entire mosquito sodium channel, which directly reflects field populations of mosquitoes that are known to naturally have multiple sodium channel mutations. This study is also unique in its characterizing the effects not only of nonsynonymous but also of synonymous mutations on sodium channel sensitivity to pyrethroids. Characterizing the roles of nonsynonymous and synonymous mutations or mutation combinations in an entire mosquito sodium channel is an important, early step in understanding molecular mechanisms involved in sodium channel insensitivity-mediated resistance and, ultimately, in informing the development of novel treatments to control mosquitoes and to prevent mosquito-borne diseases, in particular malaria. PUBLIC HEALTH RELEVANCE: The proposed project seeks to characterize sodium-channel mutations, both nonsynonymous and synonymous mutations, that are involved in pyrethroid insecticide resistance in the mosquito vector Culex quinquefasciatus. We will define the relationship between the level of insecticide resistance and prevalence of sodium channel mutations and mutation combinations in this mosquito vector. We will determine the effects of key mutations and mutation combinations on sodium channel sensitivity to pyrethroids, using the Xenopus oocyte expression system. We also will determine the post-transcriptional regulation mechanism for the key mutations in the mosquito sodium channel by comparing the alleles of each key mutation at the DNA level, with the alleles of each key mutation at the RNA level.